The Food and Drug Administration (FDA) defines cell therapy as the prevention, treatment, cure or mitigation of disease or injuries in humans by the administration of autologous, allogeneic or xenogeneic cells that have been manipulated or altered ex vivo. The goal of cell therapy, overlapping that of regenerative medicine, is to repair, replace or restore damaged tissues or organs.
Ex vivo expansion of cells obtained from human donors is being used, for example, to increase the numbers of stem and progenitor cells available for autologous and allogeneic cell therapy. For instance, multipotent mesenchymal stromal cells (MSCs) are currently exploited in numerous clinical trials to investigate their potential in immune regulation, hematopoiesis, and tissue regeneration. The low frequency of MSCs necessitates cell expansion to achieve transplantable numbers.
The challenge for any cell therapy is to assure safe and high-quality cells for transplantation, at a reasonable cost and at lot sizes able to support a commercial therapeutic product. In particular, cell processing under current Good Manufacturing Practice (cGMP)-graded conditions is mandatory for the progress of such advanced cell therapies. For allogeneic therapies, the economics of testing and certification of processes and products for cGMP compliance are a significant cost factor in cell manufacturing, strongly encouraging production of maximum batch size and minimum batch run. Importantly, cell therapies must achieve lot sizes that will supply sufficient material to meet commercial demand. Today's lot sizes of 5-20 billion cells per lot are insufficient to produce a commercial product, and lot sizes must increase to the 100 s of billions of cells yielding process volumes of 100-300 liters of cells for downstream processing. Due to inherently expensive manufacturing processes, traditional biopharmaceutical process yields of 50 percent to 70 percent are unacceptable for cell therapy products. The process economics demand lot sizes of greater than 50 billion cells and product recovery well over 80 percent if cell therapies are to be cost competitive with less complex therapeutic products such as small molecules or therapeutic proteins.
Optimally therefore, therapeutic cell manufacturing for clinical-scale expansion would be conducted in a completely automated, closed process from tissue collection through post-culture processing. Such a closed process would facilitate cGMP-compliant manufacturing of cell therapy products in a form suitable for storage and ready for use in a clinical setting, with minimal risk of microbial contamination. Some systems for such closed processes have been developed for relatively small-scale production of autologous cell therapy products (see, e.g., U.S. Pat. App. Pub. No. 2008/0175825 by Hampson et al.), but for various reasons such systems are not readily scaled for larger preparations.
Large-scale automated, closed processes for use of mammalian cells to manufacture proteins, such as biotherapeutics, are well established. However, most such processes are designed to recover a protein product and discard the cells under conditions leading to cell death, either intentionally, as when cells are disrupted for release of intracellular products, or incidentally, when cells are separated from secreted products by harsh methods such as high shear centrifugation or filtration methods. In contrast, processing of therapeutic cells after expansion typically requires cell harvesting, volume reduction, washing, formulation, filling of storage containers and, often, cryopreservation of the product cells, all under conditions maintaining cell viability, biological functionalities, safety and, ultimately, clinical efficacy.
In addition, therapeutic cells are known not to survive processes for handling cells used for protein production due to high mechanical stresses of these techniques and because the cell lines used in protein production typically represent highly-manipulated cell lines which, during extensive replication in culture, may have undergone selection for less sensitivity to mechanical shear forces and physiological stresses than exhibited, for instance, by progenitor or stem cells used in cell therapies. Thus, to retain efficacy, therapeutic cells typically are minimally cultured so as to maintain the original parental phenotype displayed upon isolation from human tissue; and hence, therapeutic cells generally are not selected or genetically engineered to facilitate downstream processing.
As technologies are developed to scale the cell culture processes, the technology required for downstream processing has quickly been overwhelmed. Specifically, volume reduction and washing of large amounts (e.g., 10-100 liters) of therapeutic cell suspensions with current technologies is time consuming and not scalable. Current technology, such as open centrifugation, may require four to eight hours by five to twenty highly trained technicians using tens to hundreds of individual processing vessels, thus increasing manipulations and risk of contamination. Much of the field of cell therapy utilizes small scale blood processing equipment, which cannot be scaled to more than about ten liters per process. Thus, processing time and labor, and production costs are major constraints to be resolved in therapeutic cell volume reduction and washing, and there are further benefits to process equipment that can scale from the five to ten liter range to several hundred liters, while at the same time maintaining the critical quality parameters of the process and resulting cell product. Such critical quality parameters include: cell suspension densities sufficient for therapeutic formulations (e.g., greater than ten million cells/ml in most cases, and at least 30-70 million cells/ml in some cases): high viability of the final cell product to maintain functionality and safety: high yield of cells to minimize loss of the high value cells; and reduction of residual levels of harvest reagents (e.g., trypsin or other enzyme) and media components (e.g., serum components, active growth factors, and the like) to acceptable levels for regulatory purposes.
Accordingly, there is a need for improved processes for manufacturing therapeutic cells, from cell collection through post-culture processing, including processes for efficient volume reduction and washing of cell suspensions with high yields of viable cells and low residual levels of culture or processing components that are detrimental to therapeutic use of the cells, particularly such processes that facilitate manufacturing in automated, closed systems.
The expansion and recovery of therapeutic cells in scalable culture system therefore requires the use of a well-regulated process that minimizes the risk of contamination, prevents product degradation and maintains product functionality while delivering cells at high concentrations and high purity for ease and efficiency during product processing. High purity cell products are important because they consist of human cells that are intended for implantation, transplantation or infusion into a human patient that must meet specific criteria to be used as therapeutics. A typical manufacturing process for cell-based therapy involves production of large scale cells, which are further recovered with high viability, high purity and of high concentration for cryopreservation in high doses before delivery to end users. Typically high viability means greater than 90 percent viable cells at this stage; however, greater than 80 percent is seen as acceptable. High purity is generally considered less than one ppm process residuals as guided by the Code of Federal Regulations (21 CFR § 610.15(b)). The challenges of therapeutic cells vs proteins and related difficulties in scale-up are further outlined in Brandenberger et al., Cell Therapy Bioprocessing Integrating Process and Product Development for the Next Generation of Biotherapeutics, BioProcess International, March 2011: 31-37; and Rowley J A, Developing Cell Therapy Biomanufacturing Processes, Chem. Eng. Progr. (SBE Stem Cell Engineering Supplement) November 2010: 50-55.
Thus, one of the main challenges in cell bioprocess technology is to manufacture and process large number of cells to satisfy the demand for lot sizes of up to 5000 doses per lot, with doses ranging from 20 million to 1 billion cells per dose. This necessitates lot sizes of 20 billion cells for low dose products to up to several trillion cells per lot for high dose indications. As cell bioprocesses have a formulation stage where formulation buffers are used to dilute cells to specific dose concentrations in the presence of biopreservative reagents (such as DMSO), cells must be at greater than final concentration prior to the formulation densities, requiring in-process cell concentrations of 0.5-2 fold above final concentrations. These specifications require a downstream technology that is able to concentrate cells up to 10-80 million cells/ml. While it is possible to further concentrate cells after separation, it is not desirable as each additional processing step leads to 5-15 percent loss of cells. If one could achieve high cell concentrations directly post-separation, overall process recoveries would be much higher, thereby achieving greater process economics.
Counterflow centrifugation separation technology is now available such as kSep® commercialized by kSep Systems Corporation. This device provides counterflow centrifugation for the concentration and washing of therapeutic cells. Counter flow centrifugation has been around since the 1940s, and is used in commercial devices such as Elutra, (sold by Caridian BCT) used in cell processing. Exemplary patents related to this technology include U.S. Pat. Nos. 5,622,819; 5,821,116; 6,133,019; 6,703,219; 6,916,652; 7,549,956; 6,214,617; 5,674,173; 4,939,087; US20110207225 and US20110207222. Counterflow centrifugation separation technology such as kSep® operates continuously and retains heavier/denser materials such as cells, while removing supernatant by net force balance from centrifugation and fluid flowrate. The cells remain in suspension during the process. Advantages include low cell shear stress and continuous supply of oxygen and nutrient rich cell suspension which keep the cells nurtured throughout the process.
However, the cell recovery for these systems is about 78 percent at approximately 60 ml/minute normal (processing) flow rate. Lowering the normal flow can increase cell recovery. Problematically, processing at lower flow rates increases the processing time to complete a harvest of about 30 liters to greater than six hours.
There is a need for a system that can process large volume batches in a reasonable time with high recovery, concentration, and product quality. There is a further need for a system that is temperature regulated, completely closed, fully disposable and scalable and includes integrated disposables designed for both the input cells and output cells (capturing waste media and processing buffer, collecting cells, and taking cells into the next processing steps). There is a further need for a system that can process (separate, clarify, recover and collect cells from the fluid media) 20-120 liters of harvested cells in less than six hours, and in alternative embodiments in less than four hours, and routinely recover greater than 85 percent of cells processed, all while maintaining high cell viability (greater than 85 percent), purity (less than 1 ppm bovine serum albumin (BSA)) and cell functionality.